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| Status |
Public on Mar 05, 2024 |
| Title |
BMMC, PBS, H3K4me1, rep1 |
| Sample type |
SRA |
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| Source name |
Cultured primary bone marrow monocytes
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| Organism |
Mus musculus |
| Characteristics |
cell type: Cultured primary bone marrow monocytes
strain: C57Bl/6J
age: 8-weeks-old
Sex: Female
treatment: PBS
antibody: H3K4me1 (EpiCypher #13-0057)
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| Treatment protocol |
BMMCs were cultured for 5 days under continuous high-dose 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) stimulation or PBS control conditions
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| Growth protocol |
In vitro bone marrow monocyte (BMMC) LPS exhaustion experiments were performed as previously described (Pradhan et al., 2021, Front. Immunol.). Briefly, bone marrow cells were harvested from 6- to 8-week-old C57BL/6 WT female mice, seeded at a density of 3x105 cells/cm2, and cultured in complete RPMI 1640 media (10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine) supplemented with 10 ng/mL M-CSF (PeproTech). Cells were cultured for 5 days at 37˚C in a humidified 5% CO2 atmosphere under continuous high-dose 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) stimulation or PBS control conditions, with fresh media changes at days 2 and 4 of culture.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cultured BMMCs were harvested by manual disruption and immediately processed for CUT&RUN using a CUTANA ChIC/CUT&RUN Kit (EpiCypher) according to the manufacturer’s protocol. Briefly, live cells were permeabilized in 0.01% digitonin in the presence of ConA beads. K-MetStat Panel spike-in was added to each reaction, and samples were incubated overnight at 4˚C in the presence of H3K4me1 (EpiCypher #13-0057), H3K4me3 (EpiCypher #13-0041), or IgG negative control (EpiCypher #13-0042) antibodies. Following PAG-MNase digestion and chromatin release, 0.5ng of E. coli spike-in DNA were added to each reaction, and DNA samples were purified using CUTANA kit reagents.
Purified DNA was quantified using a Qubit dsDNA HS Assay Kit (Invitrogen), and 1-5 ng of purified DNA were used as input for CUTANA CUT&RUN Library Prep Kit (EpiCypher) according to the manufacturer’s protocol. Library fragment sizes were analyzed using Tapestation DNA ScreenTape analysis (Agilent) at the Fralin Life Sciences Institute at Virginia Tech Genomics Sequencing Center.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 1000 |
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| Data processing |
Paired-end CUT&RUN reads were mapped to the mm10 and E. coli (MG1655) genomes using bowtie2 (2.5.2; parameters: --local –very-sensitive –no-mixed –no-discordant –dovetail)
Low quality mapped reads (-q 30) and PCR duplicates were removed using samtools, and peak calling was performed using SEACR (parameters: norm, stringent)
CUT&RUN coverage was determined using deepTools (3.5.2; function: bamCompare), with coverage values normalized to E. coli spike-in controls
bigwig files were prepared using bedGraphToBigWig (UCSC)
Assembly: mm10
Supplementary files format and content: biwgwig, bed (SEACR formatted: <chr> <start> <end> <total signal> <max signal> <max signal region>)
Library strategy: CUT&RUN
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| Submission date |
Jan 13, 2024 |
| Last update date |
Mar 05, 2024 |
| Contact name |
Blake Alexander Caldwell |
| E-mail(s) |
blakeac@vt.edu
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| Phone |
8655997842
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| Organization name |
Virginia Tech
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| Department |
Biological Sciences
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| Lab |
Li Lab
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| Street address |
970 Washington Street SW
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| City |
Blacksburg |
| State/province |
Virginia |
| ZIP/Postal code |
24061 |
| Country |
USA |
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| Platform ID |
GPL32159 |
| Series (2) |
| GSE253201 |
Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis [CUT&RUN] |
| GSE253202 |
Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis |
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| Relations |
| BioSample |
SAMN39432667 |
| SRA |
SRX23206499 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8016296_PBS-H3K4me1-1.bw |
32.6 Mb |
(ftp)(http) |
BW |
| GSM8016296_PBS-H3K4me1-1.stringent.bed.gz |
745.9 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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